Method for inducing industrial cannabis sativa to generate female seeds through inducer

Abstract

The invention belongs to the technical field of agricultural cultivation, and particularly relates to a method for inducing industrial cannabis sativa to generate female seeds through an inducer. The method comprises the following steps: (1) preparing an inducer mixed solution; the concentration of the nano-silver particles in the inducer mixed solution is 150 mg / L to 200 mg / L, the concentration of the gibberellin is 80 mg / L to 100 mg / L, and the concentration of the surfactant is 500 mg / L to 700 mg / L; (2) selecting industrial cannabis sativa which is about 10-15 days in the flowering period and is confirmed to be female plants, spraying the whole female plants of the industrial cannabis sativa in a mist form by using the inducer mixed solution in the near evening, avoiding stems, and preferably, spraying once every 2-3 days, spraying twice in total, and meanwhile, changing the illumination time, so that the female plants of the industrial cannabis sativa are obtained. The obtained industrial hemp seeds are all female. The method disclosed by the invention is high in all-female induction rate and good in stability.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural cultivation, and in particular relates to a method for inducing industrial hemp to produce female seeds by using an inducer. Background Art

[0002] Industrial hemp is an annual herbaceous plant of the genus Cannabis in the family Cannabaceae. Its THC content is less than 0.3%. Industrial hemp is a treasure from head to toe. Its applications include textile, papermaking, food, medicine, hygiene, daily chemicals, leather, automobile, construction, decoration, packaging and other fields. Currently, the most valuable extract from industrial hemp for medical and commercial research is called cannabidiol (CBD). Studies have shown that CBD has pharmacological effects such as protecting nerves, anti-oxidation, anti-anxiety and anti-inflammatory. In addition to medical use, its application has expanded to multiple fields such as consumer goods, food, and new materials.

[0003] Since the existing industrial hemp in my country is a dioecious crop, under natural conditions, the ratio of male to female seeds produced by industrial hemp sowing is 1:1. During seed production and planting, manpower and material resources are needed to eliminate male plants. If the management is not in place, even if there is one male plant missed, it will lead to pollination of the female plant and reduce the CBD content in the female plant. CBD is mainly found in the flowers and leaves of female plants, that is, only female plants contain high levels of CBD. The CBD content in male plants and seed-bearing plants is very low. Cannabis plants need both male and female plants to reproduce.

[0004] When producing industrial hemp hybrids, male plants in the mother plant need to be manually removed. The process of emasculation lasts a long time, is labor-intensive, and is difficult to completely remove. These problems restrict the breeding of industrial hemp hybrid seeds to a certain extent. By using male-inducing inducers to force female plants to produce fertile pollen to pollinate female plants, all-female industrial hemp seeds can be obtained, and their offspring are all-female industrial hemp plants. This technology can reduce the cost of searching for and removing male plants, while avoiding the risk of male plants producing anthers to pollinate female plants, saving manpower, material resources, and other costs, and is a prerequisite for improving economic benefits and accelerating the development of the industrial hemp industry.

[0005] The patent with publication number KR1020040060528A discloses a method for producing female seeds by inducing male flowers on male and female stamens. When the female cannabis is before the flowering period, a silver nitrate (AgNO3) solution is sprayed on the selected female cannabis plant, and the female cannabis plant is induced to produce male flowers under light control. However, silver nitrate is a toxic substance and is not convenient to store. Chinese patent CN202111682460.3 discloses a male induction agent for industrial hemp, a preparation method and a use method thereof. The induction success rate of this method is more than 95%, and the plant growth state is normal and the pollen amount is large. However, the induction stability of this method is not ideal.

[0006] Therefore, there is an urgent need for a method of inducing industrial hemp to produce female seeds through an inducer. Summary of the invention

[0007] The object of the present invention is to provide a method for inducing industrial hemp to produce female seeds by using an inducer.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] A method for inducing industrial hemp to produce female seeds by an inducer, comprising the following steps:

[0010] (1) preparing an inducer mixed solution, comprising the following steps: adding nanosilver particles to distilled water in a dark environment to obtain a nanosilver particle liquid; in a dark environment, using a small amount of anhydrous ethanol to completely dissolve gibberellin, adding the gibberellin to the nanosilver particle liquid, and continuously adding a surfactant, stirring until the mixture is uniform, to obtain an inducer mixed solution;

[0011] The concentration of the nano silver particles in the inducer mixture is 150mg / L-200mg / L, the concentration of the gibberellin is 80mg / L-100mg / L, and the concentration of the surfactant is 500mg / L-700mg / L;

[0012] (2) Select industrial hemp plants that are 10 to 15 days from flowering and have been confirmed to be female, and spray the whole female industrial hemp plant with the inducer mixture in the evening in the form of mist, avoiding the stems, so that there are water droplets on the leaves but no water drops, and spray again once every 2 to 3 days, for a total of 2 times, while changing the light exposure time, to obtain industrial hemp with all-female seeds.

[0013] Chinese patent CN202111682460.3 discloses an inducing agent for industrial hemp, its preparation method and use method. During the experiment, it was found that the induction stability of the method was not ideal and the batch stability of the method was poor. In a large number of experiments, it was found that when the concentration of nanosilver particles in the inducer mixture was 150mg / L~200mg / L, the concentration of gibberellin was 80mg / L~100mg / L, and the concentration of surfactant was 500mg / L~700mg / L, within the above-mentioned ratio range, the induction stability between batches of the method can be improved. The analysis shows that a synergistic effect is formed within a specific concentration range, the induction stability is higher, and together promotes the consistency and reliability of the experimental results.

[0014] Furthermore, the light exposure time was changed to avoid light from 6 pm to 6 am.

[0015] Furthermore, the surfactant comprises an alkyl glycoside, a fatty alcohol polyoxyethylene ether and a plant-derived surfactant in a weight ratio of 1: (0.4-0.6): (1.3-1.5).

[0016] Furthermore, the preparation method of the plant-derived surfactant comprises the following steps:

[0017] (1) Peeled red beans, mung beans and black beans are mixed and crushed until they completely pass through a 40-mesh sieve to obtain a mixed powder;

[0018] (2) Mixing the mixed powder with deionized water in a weight ratio of 1:(6-8) to obtain a mixture, adding composite bacteria, performing anaerobic fermentation, sterilizing after the fermentation, adding bromelain, and performing enzymolysis at 35-38° C. for 60-80 min; after the enzyme is inactivated, adjusting the pH to 2.0-2.5 with 0.1 mol / L hydrochloric acid, adding pepsin, and performing enzymolysis at 35-38° C. for 60-80 min; after the enzyme is inactivated, obtaining a hydrolyzed protein solution;

[0019] (3) centrifuging the hydrolyzed protein solution to remove solids, and freeze-drying to obtain hydrolyzed protein amino acids;

[0020] (4) After heating lauryl alcohol until it is melted, hydrolyzed protein amino acids are added, reacted at 100-105° C. for 6-7 hours, and water is removed to obtain a plant-derived surfactant.

[0021] During the experiment, it was found that if the concentration of nanosilver particles is increased, although the induction rate can be increased, it is easy to cause abnormal plant growth. In order to improve the induction rate, the present invention attempts to add a compounded surfactant to improve the effect of the use of exogenous agents, but the result is not ideal. The present invention can improve the induction rate of the method by compounding alkyl glycosides, fatty alcohol polyoxyethylene ethers and plant-based surfactants. Analysis shows that through the compounded surfactants, APG, AEO and plant-based surfactants each have different functional characteristics. Through compounding, a synergistic effect can be formed, giving full play to their respective advantages and making up for the shortcomings of a single surfactant. The compounded surfactant system can optimize the effect of the inducer mixture of the present invention in many aspects, including wettability, permeability, emulsification, dispersibility and stability, thereby comprehensively improving the use effect of exogenous agents.

[0022] Furthermore, the addition amount of bromelain is 1000-2000 U / g of the mixture.

[0023] Furthermore, the added amount of pepsin is 2000-4000 U / g of the mixture.

[0024] Furthermore, the fermentation temperature is 30-35°C, and the fermentation time is 15-20 hours.

[0025] Furthermore, the composite bacteria include Lactobacillus plantarum, Bifidobacterium longum and Lactobacillus bulgaricus.

[0026] Furthermore, the amount of the plant lactobacillus in the mixture is 10 9 ~10 10 CFU / mL; the dosage of Bifidobacterium longum in the mixture is 10 10 ~10 11 CFU / mL; the amount of Lactobacillus bulgaricus in the mixture is 10 8 ~10 9 CFU / mL.

[0027] Furthermore, the weight ratio of peeled red beans, mung beans and black beans is 1:(1.2-1.4):(0.5-0.8).

[0028] Furthermore, the weight ratio of lauryl alcohol to hydrolyzed protein amino acids is (3.4-3.7):1.

[0029] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0030] 1. The present invention provides an inducer for inducing female industrial hemp plants into males, which is a chemical induction method. Specifically, the inducer is used to evenly spray the whole female plant twice at intervals of two days. After the plant absorbs the inducer, the female plant is forced to induce male flower buds, produce a large number of fertile anthers, and the pollen is strong and dense. The success rate of inducing males is high and stable, and the female plant is pollinated and seeded to breed all-female industrial hemp seeds. The seedlings grown by the all-female seed seedlings obtained by the present invention are all-female industrial hemp plants, which are genetically stable and have an all-female rate of 99.9%. The induction cost is reduced, the procedure is simplified, and it is easy to operate.

[0031] 2. The present invention induces female plants to produce male flowers by reducing the ethylene level in female plants or inhibiting the production of ethylene. The inducer used in the present invention is non-toxic and non-corrosive, meets the requirements of environmental protection and green, and has simple configuration, low cost, and a simple and easy-to-operate induction process.

[0032] 3. When the concentration of nanosilver particles in the inducer mixture is 150 mg / L to 200 mg / L, the concentration of gibberellin is 80 mg / L to 100 mg / L, and the concentration of surfactant is 500 mg / L to 700 mg / L, within the above ratio range, the induction stability between batches of the method can be improved.

[0033] 4. The present invention can improve the induction rate of the method by compounding alkyl glycoside, fatty alcohol polyoxyethylene ether and plant-derived surfactant. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a schematic diagram of a normal female plant in Example 1;

[0035] Figure 2 This is a schematic diagram of the feminized male plant that can be pollinated in Example 1;

[0036] Figure 3 This is a schematic diagram of the state of the female plant after pollination in Example 1;

[0037] Figure 4 This is a schematic diagram of the fruiting of industrial hemp after feminization in Example 1;

[0038] Figure 5 This is a schematic diagram of seeds harvested after industrial hemp is feminized and set seeds in Example 1;

[0039] Figure 6 This is a schematic diagram of the germination of seeds harvested after industrial hemp was feminized and set seeds in Example 1. DETAILED DESCRIPTION

[0040] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] The raw materials used in the following examples of the present invention are all commercially available products:

[0042] Test variety: Longda 7.

[0043] Lactobacillus plantarum was purchased from China Center for Type Culture Collection, strain number: CCTCCAB2010210.

[0044] Lactobacillus bulgaricus was purchased from China Center for Type Culture Collection, strain number: CCTCCCB20082295.

[0045] Bifidobacterium longum was purchased from China Center for Type Culture Collection, strain number: CCTCCHB20082718.

[0046] The enzymes used were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0047] Alkyl glycosides, Dezhou Mengqiao Chemical Co., Ltd., APG 0810.

[0048] Fatty alcohol polyoxyethylene ether, AEO~9, Shandong Weilun Chemical Co., Ltd.

[0049] Example 1

[0050] This embodiment provides a method for inducing industrial hemp to produce female seeds by using an inducer, comprising the following steps:

[0051] (1) preparing an inducer mixed solution, comprising the following steps: adding nanosilver particles to distilled water in a light-proof state, stirring and mixing, to obtain a nanosilver particle dispersion; in a light-proof state, completely dissolving gibberellin in anhydrous ethanol (3 times the volume of gibberellin), adding the gibberellin to the nanosilver particle liquid, continuing to add a surfactant, and continuously stirring until the mixture is uniformly mixed, to obtain an inducer mixed solution;

[0052] The concentration of nanosilver particles in the inducer mixture was 180 mg / L, the concentration of gibberellin was 90 mg / L, and the concentration of surfactant was 600 mg / L;

[0053] (2) Select industrial hemp plants that are about 12 days from flowering and have been confirmed to be female, and spray the whole female industrial hemp plant with the inducer mixture in the form of mist at 17:00, avoiding the stems, so that there are water droplets on the leaves but no water drops, and spray again once every 3 days, for a total of 2 sprays, and at the same time, change the light exposure time, which is changed from 6 pm to 6 am to avoid light, to obtain industrial hemp with all-female seeds.

[0054] The surfactant comprises alkyl glycoside, fatty alcohol polyoxyethylene ether and plant-derived surfactant in a weight ratio of 1:0.5:1.4.

[0055] The preparation method of the plant-derived surfactant comprises the following steps:

[0056] (1) Peeled red beans, mung beans and black beans are mixed in a weight ratio of 1:1.3:0.6, and crushed until they completely pass through a 40-mesh sieve to obtain a mixed powder;

[0057] (2) Mix the mixed powder with deionized water in a weight ratio of 1:7 to obtain a mixture, and add composite bacteria, wherein the composite bacteria includes Lactobacillus plantarum, Bifidobacterium longum and Lactobacillus bulgaricus. The amount of Lactobacillus plantarum in the mixture is 10 9 CFU / mL; the dosage of Bifidobacterium longum in the mixture is 10 11 CFU / mL; the amount of Lactobacillus bulgaricus in the mixture is 10 8 CFU / mL; perform anaerobic fermentation at a fermentation temperature of 32°C for 18 hours; sterilize after the fermentation, add bromelain at a rate of 1500U / g of the mixture, and perform enzymolysis at 36°C for 70 minutes; after inactivating the enzyme, adjust the pH to 2.5 with 0.1mol / L hydrochloric acid, add pepsin at a rate of 3000U / g of the mixture, and perform enzymolysis at 36°C for 70 minutes; after inactivating the enzyme, obtain a hydrolyzed protein solution;

[0058] (3) centrifuging the hydrolyzed protein solution to remove solids, and freeze-drying to obtain hydrolyzed protein amino acids;

[0059] (4) After heating lauryl alcohol until it is melted, hydrolyzed protein amino acids are added, and the weight ratio of lauryl alcohol to hydrolyzed protein amino acids is 3.5:1; reacting at 102° C. under stirring for 6.5 hours, and removing water by reduced pressure rotary evaporation to obtain a plant-based surfactant.

[0060] Example 2

[0061] The difference between this embodiment and embodiment 1 is that the concentration of the nanosilver particles in the inducer mixture is 150 mg / L, the concentration of the gibberellin is 100 mg / L, and the concentration of the surfactant is 500 mg / L.

[0062] Example 3

[0063] The difference between this embodiment and embodiment 1 is that the concentration of the nanosilver particles in the inducer mixture is 200 mg / L, the concentration of the gibberellin is 80 mg / L, and the concentration of the surfactant is 700 mg / L.

[0064] Comparative Example 1

[0065] The difference between this comparative example and Example 1 is that the concentration of the nanosilver particles in the inducer mixture is 300 mg / L, the concentration of the gibberellin is 30 mg / L, and the concentration of the surfactant is 1000 mg / L.

[0066] Comparative Example 2

[0067] The difference between this comparative example and Example 1 is that the concentration of nanosilver particles in the inducer mixture is 280 mg / L, the concentration of gibberellin is 190 mg / L, and the concentration of surfactant is 400 mg / L.

[0068] Comparative Example 3

[0069] The difference between this comparative example and Example 1 is that the surfactant is an alkyl polyglycoside.

[0070] Comparative Example 4

[0071] The difference between this comparative example and Example 1 is that the surfactant comprises alkyl glycoside, fatty alcohol polyoxyethylene ether and plant-derived surfactant in a weight ratio of 1:1:1. The preparation method of the plant-derived surfactant is the same as that of Example 1.

[0072] Comparative Example 5

[0073] The difference between this comparative example and Example 1 is that the surfactant comprises alkyl glycoside, fatty alcohol polyoxyethylene ether and silicone in a weight ratio of 1:0.5:1.4.

[0074] Comparative Example 6

[0075] The difference between this comparative example and Example 1 is that the peeled red beans, mung beans and black beans are replaced by an equal amount of soybeans.

[0076] Performance Testing

[0077] The methods of Examples 1 to 3 and Comparative Examples 1 to 6 were used for cultivation, with 100 plants in each group. Three parallel groups were set for each Example (Comparative Example). The effects of each treatment on the female plants were investigated after 15 days of treatment. The results are shown in Tables 1 to 3.

[0078] Table 1 Number of successfully induced male plants in the first group of treatments

[0079] Each treatment Drug damage Number of successful male induced plants Plant performance Example 1 none 99 Normal growth status Example 2 none 98 Normal growth status Example 3 none 97 Normal growth status Comparative Example 1 none 93 Normal growth status Comparative Example 2 none 90 Normal growth status Comparative Example 3 none 94 Normal growth status Comparative Example 4 none 92 Normal growth status Comparative Example 5 none 95 Normal growth status Comparative Example 6 none 94 Normal growth status

[0080] Table 1 Number of successfully induced male plants in the second group of treatment

[0081] Each treatment Drug damage Number of successful male induced plants Plant performance Example 1 none 99 Normal growth status Example 2 none 99 Normal growth status Example 3 none 99 Normal growth status Comparative Example 1 none 90 Normal growth status Comparative Example 2 none 91 Normal growth status Comparative Example 3 none 90 Normal growth status Comparative Example 4 none 91 Normal growth status Comparative Example 5 none 96 Normal growth status Comparative Example 6 none 92 Normal growth status

[0082] Table 1 Number of successfully induced male plants in the third group of treatments

[0083] Each treatment Drug damage Number of successful male induced plants Plant performance Example 1 none 99 Normal growth status Example 2 none 99 Normal growth status Example 3 none 99 Normal growth status Comparative Example 1 none 91 Normal growth status Comparative Example 2 none 90 Normal growth status Comparative Example 3 none 94 Normal growth status Comparative Example 4 none 95 Normal growth status Comparative Example 5 none 90 Normal growth status Comparative Example 6 none 91 Normal growth status

[0084] In summary, the use of the male nano-silver particles for industrial hemp in Examples 1 to 3 of the present invention to induce male hemp in female industrial hemp plants has a high induction success rate, good method stability, produces a large amount of fertile pollen, has strong pollen vitality and dense eyes, and the plant growth state is normal. Figures 1 to 6 It can be seen that the plant growth state of Example 1 is good, the induction rate can reach 99%, and the stability is excellent.

[0085] However, the comparative examples are significantly inferior to the embodiments in the corresponding performance tests because they do not adopt the necessary technical solutions. In comparative examples 1 to 2, the ratio of each component in the inducer mixture is changed, and it can be seen that the induction rate stability effect is reduced, and the stability between batches is poor. The composition of the surfactants in comparative examples 3 to 5 is different, and it can be seen that the induction rate is reduced. In comparative example 6, soybeans are used to prepare plant-based surfactants, and the induction rate is reduced. The analysis is that different types of beans contain different amino acid compositions, and different types of beans have different protein structure complexities. The composite bacteria used may have different effects on different types of beans, so the obtained plant-based surfactants are different. Only the plant-based surfactants prepared by red beans, mung beans and black beans, combined with the other two, can obtain a good wetting effect. The above experimental results further prove the importance of the technical solution defined in the present invention for its technical effect.

[0086] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for inducing industrial hemp to produce female seeds by an inducer, characterized in that: The following steps are involved: (1) preparing an inducer mixed solution, comprising the following steps: adding nanosilver particles to distilled water in a dark environment to obtain a nanosilver particle liquid; dissolving gibberellin completely in anhydrous ethanol in a dark environment, adding the gibberellin to the nanosilver particle liquid, and continuously adding a surfactant, stirring until the mixture is uniform, to obtain an inducer mixed solution; The concentration of the nano silver particles in the inducer mixture is 150mg / L-200mg / L, the concentration of the gibberellin is 80mg / L-100mg / L, and the concentration of the surfactant is 500mg / L-700mg / L; (2) Select industrial hemp plants that are 10 to 15 days from flowering and have been confirmed to be female, and spray the whole female industrial hemp plant with the inducer mixture in the evening in the form of mist, avoiding the stems, so that there are water droplets on the leaves but no water drops, and spray again once every 2 to 3 days, for a total of 2 times, while changing the light exposure time, to obtain industrial hemp with all-female seeds.

2. The method for inducing industrial hemp to produce female seeds by an inducer according to claim 1, characterized in that: The surfactant comprises an alkyl glycoside, a fatty alcohol polyoxyethylene ether and a plant-derived surfactant in a weight ratio of 1: (0.4-0.6): (1.3-1.5).

3. The method for inducing industrial hemp to produce female seeds by an inducer according to claim 2, characterized in that: The preparation method of the plant-derived surfactant comprises the following steps: (1) Peeled red beans, mung beans and black beans are mixed and crushed until they completely pass through a 40-mesh sieve to obtain a mixed powder; (2) Mixing the mixed powder with deionized water in a weight ratio of 1:(6-8) to obtain a mixture, adding composite bacteria, performing anaerobic fermentation, sterilizing after the fermentation, adding bromelain, and performing enzymolysis at 35-38° C. for 60-80 min; after the enzyme is inactivated, adjusting the pH to 2.0-2.5 with 0.1 mol / L hydrochloric acid, adding pepsin, and performing enzymolysis at 35-38° C. for 60-80 min; after the enzyme is inactivated, obtaining a hydrolyzed protein solution; (3) centrifuging the hydrolyzed protein solution to remove solids, and freeze-drying to obtain hydrolyzed protein amino acids; (4) After heating lauryl alcohol until it is melted, hydrolyzed protein amino acids are added, reacted at 100-105° C. for 6-7 hours, and water is removed to obtain a plant-derived surfactant.

4. The method for inducing industrial hemp to produce female seeds by an inducer according to claim 3, characterized in that: The added amount of bromelain is 1000-2000U / g of mixed material.

5. The method for inducing industrial hemp to produce female seeds by an inducer according to claim 4, characterized in that: The amount of pepsin added is 2000-4000U / g of mixed material.

6. The method of inducing industrial hemp to produce female seeds by an inducer according to claim 5, characterized in that: The fermentation temperature is 30-35°C and the fermentation time is 15-20 hours.

7. The method of inducing industrial hemp to produce female seeds by an inducer according to claim 6, characterized in that: The composite bacteria include Lactobacillus plantarum, Bifidobacterium longum and Lactobacillus bulgaricus.

8. The method of inducing industrial hemp to produce female seeds by using an inducer according to claim 7, characterized in that: The amount of plant lactobacillus in the mixture is 10 9 ~10 10 CFU / mL; the dosage of Bifidobacterium longum in the mixture is 10 10 ~10 11 CFU / mL; the amount of Lactobacillus bulgaricus in the mixture is 10 8 ~10 9 CFU / mL.

9. The method of inducing industrial hemp to produce female seeds by using an inducer according to claim 3, characterized in that: The weight ratio of peeled red beans, mung beans and black beans is 1:(1.2-1.4):(0.5-0.8).

10. The method for inducing industrial hemp to produce female seeds by using an inducer according to claim 3, characterized in that: The weight ratio of lauryl alcohol to hydrolyzed protein amino acids is (3.4-3.7):1.

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